Balanced Ero1 activation and inactivation establishes ER
redox homeostasis
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Citation
Kim, S. et al. “Balanced Ero1 Activation and Inactivation
Establishes ER Redox Homeostasis.” The Journal of Cell Biology
196.6 (2012): 713–725. Web. 4 May 2012.
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http://dx.doi.org/10.1083/jcb.201110090
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JCB: Article
Published March 12, 2012
Balanced Ero1 activation and inactivation
establishes ER redox homeostasis
Sunghwan Kim, Dionisia P. Sideris, Carolyn S. Sevier, and Chris A. Kaiser
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was
inactivated by both autonomous oxidation and Pdi1pmediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative
levels of reduced and oxidized glutathione in the ER to
control Ero1p activity and ensure that cells generate the
minimum number of disulfide bonds needed for efficient
oxidative protein folding.
Introduction
A key step in the maturation of many secreted proteins and extra­
cellular domains of membrane proteins is the formation of di­
sulfide bonds in the ER. Disulfide bonds, which stabilize native
and functional conformations of proteins, are formed by pairing
and oxidation of cysteines during the initial folding process in
the ER. In Saccharomyces cerevisiae, most biosynthetic disul­
fide bonds are formed by dithiol/disulfide transfer reactions
with the oxidized form of protein disulfide isomerase (PDI),
Pdi1p (Sevier and Kaiser, 2002; Wilkinson and Gilbert, 2004).
Pdi1p, in turn, requires other oxidizing molecules to be recycled
because Pdi1p cannot generate disulfide bonds by itself. We
and other colleagues identified the major disulfide-generating
flavoenzyme Ero1p in S. cerevisiae (Frand and Kaiser, 1998;
Pollard et al., 1998). In both yeast and mammalian cells, Ero1
directly transfers disulfide bonds to PDI (Frand and Kaiser, 2000;
Mezghrani et al., 2001). The oxidative capacity of the yeast ER
depends primarily on the activity of Ero1p: a temperature-sensitive
ero1-1 mutation decreases the resistance of yeast to the reducing
agent DTT and induces the unfolded protein response with ac­
cumulation of secretory proteins, and, after prolonged incubation
Correspondence to Chris A. Kaiser: ckaiser@mit.edu
C.S. Sevier’s present address is Dept. of Molecular Medicine, Cornell University,
Ithaca, NY 14853.
Abbreviations used in this paper: BSO, buthionine sulfoximine; CPY, carboxypeptidase Y; FAD, flavin adenine dinucleotide; MBP, maltose binding protein;
PDI, protein disulfide isomerase; SMM, supplemented minimal medium; TEV,
Tobacco etch virus.
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 196 No. 6 713–725
www.jcb.org/cgi/doi/10.1083/jcb.201110090
at the restrictive temperature, a strain with this mutation loses
viability (Frand and Kaiser, 1998, 1999; Cuozzo and Kaiser, 1999;
Tu and Weissman, 2002), and overexpression of ERO1 from
a multicopy plasmid increases the oxidizing capacity of yeast
cells, as shown by their increased resistance to DTT (Frand and
Kaiser, 1998).
The catalytic cycle of Ero1p depends on a relay of disulfide
bonds from the conserved active-site cysteine pair, C352-C355,
which is proximal to bound flavin adenine dinucleotide (FAD)
to the second shuttle cysteine pair, C100-C105, which is respon­
sible for direct disulfide transfer to Pdi1p (Frand and Kaiser,
2000; Gross et al., 2004; Sevier and Kaiser, 2006b). In addition
to these two catalytic cysteine pairs, Ero1p contains three cyste­
ine pairs (C90-C349, C143-C166, and C150-C295) that form
regulatory bonds (Sevier et al., 2007). Several lines of evidence
indicate that Ero1p is inactive with regulatory bonds formed,
whereas it is active with regulatory cysteines in the reduced
state. Ero1p is converted from an oxidized (inactive) state to the
reduced (active) state in the presence of reduced thioredoxin-1
(Trx1) as a substrate; once Trx1 is fully oxidized through the
action of active Ero1p, Ero1p returns to the oxidized (inactive)
form (Sevier et al., 2007). A double mutant, Ero1p-C150A-C295A,
which cannot form the crucial C150-C295 regulatory bond,
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THE JOURNAL OF CELL BIOLOGY
T
he endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through
the action of Ero1p, which generates disulfide bonds,
and Pdi1p, which receives disulfide bonds from Ero1p
and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory
bonds within Ero1p is essential for maintaining the proper
redox balance in the ER. In this paper, we show that Pdi1p
is the key regulator of Ero1p activity. Reduced Pdi1p resulted
in the activation of Ero1p by direct reduction of Ero1p
© 2012 Kim et al. This article is distributed under the terms of an Attribution–Noncommercial–
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Supplemental Material can be found at:
http://jcb.rupress.org/content/suppl/2012/03/07/jcb.201110090.DC1.html
http://jcb.rupress.org/content/suppl/2012/04/02/jcb.201110090.DC2.html
JCB
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Published March 12, 2012
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Figure 1. Ero1p regulation by reduction or oxidation of its regulatory
bonds. (A) Disulfide connectivity of Ero1p and Pdi1p. In Ero1p, dark gray
and light gray circles indicate catalytic and regulatory cysteines, respectively. Pdi1p has four thioredoxin domains, a, b, b, and a. In Pdi1p,
dark gray and black circles indicate catalytic and structural cysteines,
respectively. (B) Ero1p cycles between oxidized (ox) and reduced (red)
regulatory states in vivo according to the redox environment. Cells were
incubated with 0, 0.2, or 1 mM DTT for 30 min. DTT was removed by
placing cells in fresh medium. (C) Ero1p regulation in vitro. 1 µM Ero1pc
was incubated with 50 µM reduced Trx1.
et al., 2008). We have reconstituted this coupled reaction in vitro
and show that it recapitulates properties of the ER in vivo; as
long as an excess of reduced glutathione is available, Pdi1p is
maintained in a state enabled to activate Ero1p, which in turn
provides oxidizing equivalents to oxidize glutathione.
Results
Ero1p is capable of autonomous
inactivation
Because the regulatory bonds link distant sites of the Ero1p
polypeptide chain, the presence or absence of these bonds can
be readily discerned by mobility differences on nonreducing
SDS-PAGE (Fig. 1 A). Activity of Ero1p can be inferred from
the redox state of the regulatory bonds. In live yeast cells, Ero1p
is converted to the reduced (active) form in cells treated with
the reducing agent DTT and then returns to the oxidized (inactive)
form after DTT removal (Fig. 1 B). Activation of Ero1p by re­
duction of the regulatory bonds and subsequent inactivation
by reformation of the regulatory bonds could also be readily
observed in an in vitro assay containing recombinant Ero1p
(Ero1pc, 56–424, with an N-terminal (1–55) and a C-terminal
(425–563) fragment removed) and an excess of reduced Trx1,
Downloaded from jcb.rupress.org on April 18, 2012
exhibits increased Ero1p activity in vitro and in vivo (Sevier
et al., 2007).
A byproduct of Ero1p activity is the generation of hydro­
gen peroxide as a result of a two-electron reduction of oxygen
per disulfide generated. Thus, although Ero1p activity is essential
for biosynthetic disulfide bond formation, uncontrolled Ero1p
activity could produce too much reactive oxygen species that
would be detrimental to the cell (Gross et al., 2006). Indeed,
overexpression of the hyperactive Ero1p-C150A-C295A mutant
inhibits cell growth, highlighting the physiological importance
of the regulatory bonds in keeping Ero1p activity in check.
Mammalian Ero1- has been shown to modulate its activity
through a similar mechanism involving regulatory bonds, show­
ing that autoregulation in response to the redox environment is a
general property of Ero1 (Appenzeller-Herzog et al., 2008; Baker
et al., 2008). An additional layer of protection against reactive
oxygen accumulation in the ER of mammalian cells is provided by
an ER resident, peroxiredoxin, which reduces hydrogen peroxide
generated by Ero1 to limit peroxide accumulation (Tavender et al.,
2008, 2010; Zito et al., 2010). An obvious sequence homolog to
peroxiredoxin is absent in yeast; whether additional pathways
functionally similar to the peroxiredoxin system exist in the ER of
fungi remains to be explored.
The precise mechanism by which the regulatory bonds of
Ero1 are reduced and then reoxidized will be crucial to under­
stand how Ero1p activity is controlled and which features of the
redox environment of the ER are sensed by Ero1p. Pdi1p is the
most abundant oxidoreductase of the yeast ER and is the pre­
ferred physiological substrate for oxidation by Ero1p (Nørgaard
et al., 2001; Vitu et al., 2010); therefore, Pdi1p is likely to play
an important part in regulating Ero1p. In the mammalian ER,
the disulfide relay between Ero1-a and PDI is regulated at least
in part by the availability of PDI (Appenzeller-Herzog et al.,
2010; Inaba et al., 2010). Interaction between Ero1- and PDI
has been shown to be facilitated by a protruding -hairpin
in Ero1- and the b domain of PDI, which facilitates the inter­
conversion between the Ox1 and Ox2 redox-active forms of
Ero1- (Masui et al., 2011). Differences between Ero1- and
Ero1p, including the lack of a -hairpin in Ero1p and the prefer­
ence of Ero1p for the a domain of Pdi1p (versus the preference of
Ero1- for the a domain), suggest that the yeast and mam­
malian Ero1-PDI systems may operate differently.
In vitro assays showing activation of Ero1p have typically
used the nonphysiological substrate Trx1, which has a single
CxxC-containing domain with a highly reducing reduction po­
tential rather than the physiological substrate Pdi1p because re­
duced Trx1 is more effective than Pdi1p at activating Ero1p by
reduction of regulatory bonds (Sevier et al., 2007; Baker et al.,
2008; Vitu et al., 2010). Here, we examine the role of Pdi1p in the
reduction and reoxidation of the Ero1p regulatory bonds to
reveal how the activity of Ero1p is set in a living cell. We have also
sought to understand the role of glutathione in this process as the
most abundant redox buffer of the ER. Although glutathione
itself does not activate Ero1p nor is glutathione efficiently used as
a substrate for Ero1p (Tu et al., 2000; Sevier and Kaiser, 2006b),
reduced glutathione can stimulate Ero1p activity when used in
conjunction with Pdi1p as a substrate (Sevier et al., 2007; Baker
Published March 12, 2012
Figure 2. Pathways for Ero1p inactivation. (A) To
test the ability of Ero1p to oxidize itself autonomously, 1 µM reduced (red) Ero1pc was prepared
by incubation with 100 µM reduced His6-tagged
Trx1 for 2 min and then passage through Ni2+ beads
to remove Trx1. Trx1 depletion was confirmed by
SDS-PAGE (Mix; initial mixture: Ni2+-BF [beads
bound fraction] and Ni2+-UF [unbound fraction]).
(B) Autonomous reoxidation (ox) of Ero1pc after
removal of Trx1 occurs rapidly and efficiently but
depends on a functional active site (C355), and efficient reoxidation depends on the shuttle disulfide
(C100-C105). The first lane in each time course
() shows the oxidation state of Ero1pc before reduction by Trx1. (C) Ero1pc, expressed as an MBP
fusion (MBP-Ero1pc), can reoxidize Ero1pc-C355S
in trans. An equal mixture of MBP-Ero1pc (or MBPero1-C100A-C105A; right) and Ero1pc-C355S
was reduced by incubation with reduced Trx1
followed by Trx1 depletion. The time course of reoxidation of MBP-Ero1pc and Ero1pc-C355S was followed independently by separating MBP-Ero1pc and
Ero1pc-C355S by size in SDS-PAGE and immu­no­
blotting for MBP or Ero1.
Downloaded from jcb.rupress.org on April 18, 2012
serving as an effective substrate for Ero1p (Fig. 1 C). First,
Ero1pc is inactive with the regulatory bonds in a fully oxidized
state. In 0.5 min after addition of reduced Trx1, the regulatory
bonds of Ero1pc become reduced, and Ero1pc is thus activated
for the oxidation of Trx1. As a consequence of Ero1pc, after
60 min, all of the Trx1 is oxidized, and Ero1pc returns to the
oxidized inactive form (Fig. 1 C).
Activation of Ero1pc by reduction of the regulatory bonds
initiates on addition of reduced Trx1, indicating that the regu­
latory bonds can be reduced by direct dithiol/disulfide ex­
change with reduced Trx1. For the end phase of the reaction in
which most of the Trx1 is oxidized and the regulatory bonds of
Ero1pc become reoxidized, we wished to know whether reoxi­
dation of the regulatory bonds occurred by direct oxidation by
oxidized Trx1 or instead by autonomous reoxidation by Ero1p
when all of the reduced Trx1 had been consumed. To distin­
guish between these possibilities, we first tested the ability of
active Ero1pc to oxidize regulatory cysteines in the absence of
Trx1. For this experiment, 1 µM Ero1pc was incubated with
100 µM His6-tagged reduced Trx1 for 2 min, a time sufficient
for complete activation of Ero1pc by reduction of the regula­
tory bonds. Subsequently, His6-tagged Trx1 was removed by
affinity to Ni2+ beads (Fig. 2 A). Trx1 depletion was confirmed
by the significant absence of Trx1 in the Ni2+ unbound fraction
and corresponding presence of Trx1 bound to the Ni2+ beads
(unbound faction; Fig. 2 A, bottom). A small amount of residual
Trx1 was observed in the unbound fraction (Fig. 2 A, bottom),
but it was anticipated that this limited fraction of Trx1 would
be rapidly oxidized by Ero1pc and not contribute to our Ero1pc
inactivation assay. After depletion of reduced Trx1, Ero1pc
was converted from a fully reduced to an oxidized form over a
period of 60 min with a half time for completion of 10 min
(Fig. 2 B, top). The bands migrating between the fully reduced
and oxidized forms likely correspond to partially oxidized
states of Ero1pc. Parallel reactions using Ero1pc mutants lack­
ing the shuttle disulfide bond (Ero1pc-C100A-C105A) or the
catalytic disulfide bond (Ero1pc-C355S) in the absence of Trx1
exhibited greatly slowed or no autonomous reoxidation of
Ero1pc, respectively (Fig. 2 B, middle and bottom). In addition,
when the reoxidation reaction was performed in the absence of
O2 as an electron acceptor (Fig. S1 A, right), Ero1pc exhibited
slowed autonomous reoxidation. These observations show that
Ero1pc is capable of autonomous oxidation to form regulatory
bonds and that this oxidation has catalytic requirements similar
to biosynthetic disulfide bond formation.
We considered the possibility that autonomous oxidation
of the Ero1p regulatory bonds was promoted by the hydrogen
peroxide generated by Ero1pc before removal of Trx1. There­
fore, we examined the effects of hydrogen peroxide. Addition
of catalase to the reaction, which should convert any hydrogen
peroxide into water and oxygen (Fig. S1 B), did not signifi­
cantly slow autonomous oxidation of the Ero1p regulatory bonds
(Fig. S1 A, middle). Conversely, addition of 100 µM hydro­
gen peroxide, the maximum concentration that could be obtained
from complete oxidation of 100 µM Trx1, did not enhance the
oxidation of Ero1pc-C100A-C105A (Fig. S1 C).
Autonomous oxidation of Ero1p occurs via
an intermolecular reaction
Autonomous oxidation of Ero1p could conceivably occur by
intramolecular (in cis) or intermolecular (in trans) disulfide bond
transfer. In the crystal structure of Ero1pc (Gross et al., 2004),
one of the regulatory bonds, C90-C349, is close enough to the
catalytic site to be oxidized in cis, whereas the other two regula­
tory bonds, C150-C295 and C143-C166, are located >20 Å away
from the catalytic site, suggesting that the oxidation of these
Redox control of Ero1 • Kim et al.
715
Published March 12, 2012
Ero1p is activated slowly but inactivated
rapidly by Pdi1p
As we have shown, reduced Trx1 is a potent activator of Ero1p,
and the use of Trx1 as an activator and substrate for Ero1p in
in vitro assays made it possible to document the dramatic effect
that reduction and oxidation of regulatory cysteines have on Ero1p
activity (Fig. 1 C; Sevier et al., 2007). However, Pdi1p is the major
physiological substrate of Ero1p (Frand and Kaiser, 1999), and
we were interested to use the in vitro assay for Ero1p regulation
to make a comparable assessment of the ability of Pdi1p to
either activate or inactivate Ero1p activity through reduction or
oxidation of regulatory bonds.
First, we tested the ability of oxidized Pdi1p to inactivate
Ero1p. We prepared activated Ero1pc-C100A-C105A by treat­
ment with reduced Trx1 followed by removal of Trx1. The use
of the Ero1pc-C100A-C105A mutant prevented efficient auton­
omous reoxidation of Ero1p, allowing us to test the ability of
exogenous agents to reoxidize Ero1p. Oxidized Pdi1p and Trx1
were prepared by air oxidation during purification from Escherichia coli. The endpoint of air oxidation yielded Pdi1p that had
the a domain almost fully oxidized and the a domain 50%
716
JCB • VOLUME 196 • NUMBER 6 • 2012
oxidized (Fig. S2) and Trx1 that was almost fully in the oxidized
form (not depicted). We found that addition of a 20-fold molar
excess of oxidized Trx1 had no effect on the rate of reoxida­
tion of Ero1pc-C100A-C105A (Fig. 3 A, top), showing that
oxidized Trx1 has little or no capability to oxidize the regula­
tory bonds of Ero1p. In contrast, oxidized Pdi1p dramatically
increased the rate of Ero1pc-C100A-C105A reoxidation (Fig. 3 A,
top right). The ability of oxidized Pdi1p to efficiently oxidize
the regulatory bonds of Ero1p was not a peculiarity of the
Ero1pc-C100A-C105A mutant, as we could also show that
oxidized Pdi1p accelerated the oxidation of wild-type Ero1pc
as well as Ero1pc-C355S, which is unable to oxidize its regula­
tory bonds (Fig. S3). These results show that Ero1p can be in­
activated by direct feedback from Pdi1p.
Next, we tested the converse process, the ability of re­
duced Pdi1p to reduce and thus activate Ero1p. To assess the
activation process selectively, we again used Ero1pc-C100AC105A because of this mutant’s greatly diminished capacity for
autonomous oxidation of regulatory cysteines once reduced
with an exogenous agent (Fig. 2 B, middle). Reduced Pdi1p re­
quires 60 min to reduce the Ero1p regulatory bonds completely,
whereas reduced Trx1 requires <5 min, indicating that Pdi1p is
a relatively poor activator of Ero1p (Fig. 3 B, top). In summary,
Trx1 is a potent activator but poor inactivator of Ero1p, whereas
Pdi1p is a weak activator but potent inhibitor of Ero1p. These
contrasting abilities to reduce or oxidize the regulatory bonds of
Ero1p can be accounted for by the much greater reducing po­
tential of Trx1 as compared with Pdi1p. By assaying the rate of
activation or inactivation of Ero1p in separate reactions, we
could show that reduced Pdi1p activates Ero1p more slowly
than oxidized Pdi1p can inactivate Ero1p. This explains why in
an in vitro Ero1p oxidation reaction that contains reduced Pdi1p
as a substrate, Ero1p rarely, if ever, becomes fully activated,
and the reaction proceeds relatively slowly.
The a and a domains of Pdi1p differ
in their ability to regulate Ero1p
Yeast Pdi1p has two redox-active thioredoxin-like domains with
different reduction potentials: the a domain (195 mV) and a
domain (164 mV; Fig. 1 A; Tian et al., 2006; Vitu et al., 2010).
We recently showed that Ero1p preferentially oxidizes the a
domain both in vivo and in vitro, suggesting Ero1p provides oxi­
dizing equivalents to the ER mainly via the a domain (Vitu et al.,
2010). Thus, the a domain may play a lesser role in the oxida­
tion of ER substrates despite its higher reduction potential than
the a domain. To fully understand the interactions between Pdi1p
and Ero1p, we wished to evaluate the contribution of each of the
active Pdi1p domains to reduction and oxidation of Ero1p regu­
latory bonds.
Recombinant Pdi1p purified from E. coli had a fully oxidized
a domain and a partially oxidized a domain (Fig. S2), consistent
with the different reduction potentials of the two domains. More­
over, naturally oxidized purified domain mutants Pdi1p-CCSS
(a domain cysteines intact) and Pdi1p-SSCC (a domain cyste­
ines intact) had similar oxidation states to the corresponding
domains in wild-type Pdi1p (unpublished data). The same assay
for Pdi1p oxidation of the Ero1p regulatory bonds was performed
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bonds would require involvement of additional dithiol/disulfide
exchanges with other cysteines. To determine whether one Ero1p
protein can oxidize the regulatory bonds of another Ero1p, we
asked whether Ero1pc-C355S, which is catalytically inactive
and is not capable of autonomous oxidation, could nevertheless
be oxidized in trans by active Ero1pc. A maltose binding pro­
tein (MBP) fusion to Ero1pc was used in this experiment so that
MBP-Ero1pc and Ero1pc-C355S could be distinguished by
their distinct mobilities on SDS-PAGE. An autonomous oxida­
tion assay was established by combining MBP-Ero1pc and
Ero1pc-C355S with reduced Trx1 to reduce the regulatory bonds
on both forms of Ero1p. After Trx1 was removed by Ni2+ affinity,
both Ero1pc-C355S and MBP-Ero1pc were reoxidized at nearly
equal rates, showing that efficient trans oxidation can occur
(Fig. 2 C). Consistent with the oxidation of Ero1pc-C355S
being a product of MBP-Ero1pc catalytic function, disruption
of the MBP-Ero1p catalytic cycle by mutating the C100-C105
pair (MBP-ero1-C100A-C105A) prevented Ero1pc-C355S oxi­
dation (Fig. 2 C). We also tested the effect of dilution of Ero1pc
in our autonomous oxidation assay and found that a fourfold
dilution of Ero1pc slowed autonomous oxidation of Ero1pc
(unpublished data). Together, the trans oxidation experiment and
the dilution experiment show that autonomous oxidation of the
Ero1p regulatory disulfides occurs predominantly, if not exclu­
sively, by one Ero1p protein oxidizing the regulatory bonds of
another Ero1p. The simplest mechanism for such dithiol/disul­
fide exchange would be for successive direct transfer of the
C100-C105 shuttle cysteine disulfide bond to each of the three
regulatory cysteine pairs. However, we cannot rule out the pos­
sibility that more complicated dithiol/disulfide rearrangements
occur. Slow oxidation of Ero1pc facilitated by the Ero1pcC100A-C105A (but not the Ero1pc-C355S) mutant could be
observed also at late time points of the autonomous oxidation
assay (>20 min), suggesting that the C352-C355 active site may
contribute directly to oxidation of Ero1pc.
Published March 12, 2012
Figure 3. Regulation of Ero1p by Pdi1p. (A) Reoxidation (ox) of Ero1pc-C100A-C105A, which is impaired
for autonomous oxidation, can be stimulated by oxidized
Pdi1p but not by oxidized Trx1. Ero1pc-C100A-C105A
was reduced (red) by incubation with reduced Trx1 followed by Trx1 depletion. Reoxidation of Ero1pc-C100AC105A was followed after addition of a 20-fold molar
excess of either oxidized Trx1 (ox-Trx1), oxidized Pdi1p
(ox-PDI), or oxidized Pdi1p variants (a mutant–ox-CCSS,
a mutant–ox-SSCC, and both the a and a mutant–SSSS).
(B) Reduced Pdi1p is capable of reducing Ero1pc-C100AC105A albeit at a slower rate than reduction by reduced
Trx1. Ero1pc-C100A-C105A was incubated with a 20fold excess of reduced Trx1 (red-Trx1), reduced Pdi1p
(red-Pdi1p), or reduced Pdi1p variants (the red-Pdi1p
panel is presented again in Fig. 4 C). (C) Oxidized Ero1pcC100A-C105A was mixed with different admixtures of
reduced and oxidized Pdi1p for 1 h (the time required for
100% reduced Pdi1p to reduce Ero1pc-C100A-C105A).
The relative amounts of reduced, partially reduced, and
oxidized Ero1pc-C100A-C105A were quantified by
band intensity.
Downloaded from jcb.rupress.org on April 18, 2012
with Pdi1p-CCSS or Pdi1p-SSCC mutants. Both Pdi1p mutants
accelerated oxidation of the Ero1p regulatory bonds. Despite
the less-active disulfides, Pdi1p-SSCC reproducibly accelerated
Ero1p oxidation more rapidly than Pdi1p-CCSS (Fig. 3 A), con­
sistent with the higher reduction potential of the a CxxC.
A Pdi1p-SSSS that lacked both redox-active sites did not acceler­
ate oxidation of Ero1p regulatory bonds to the extent of the
other Pdi1p variants, but a modest acceleration of Ero1p oxida­
tion was observed (Fig. 3 A, bottom right). It is possible that the
structural (CX6C) disulfide in Pdi1p (Fig. 1 A) has some effect
or that Pdi1p may stabilize the oxidized form of Ero1p through
a mechanism not involving disulfide exchange with the redoxactive motifs.
Next, we compared the ability of reduced Pdi1p-CCSS or
Pdi1p-SSCC to reduce the regulatory bonds of Ero1pc-C100AC105A. Although reduction potentials measured against a glu­
tathione standard suggest that the a domain is a better reductant,
both reduced Pdi1p-CCSS and reduced Pdi1p-SSCC reduced
Ero1p regulatory bonds at similar rates (Fig. 3 B, bottom). As
expected, the single-domain mutants reduced the Ero1p regula­
tory bonds somewhat less efficiently than did wild-type Pdi1p,
which has both active sites intact, and the double domain
mutant Pdi1p-SSSS did not reduce the Ero1p regulatory bonds
(Fig. 3 B, bottom right).
The redox state of Ero1p can be set by a
mix of reduced and oxidized Pdi1p
Reduced and oxidized Pdi1p promote activation and inactivation
of Ero1p, respectively, suggesting that the activity of Ero1p in
steady state may be set by the ratio of reduced to oxidized Pdi1p. By
taking advantage of Ero1pc-C100A-C105A, which lacks the abil­
ity to oxidize Pdi1p efficiently, we could test admixtures of
oxidized and reduced Pdi1p for their effect on the steady-state
redox balance of the regulatory bonds of Ero1pc-C100A-C105A.
Redox control of Ero1 • Kim et al.
717
Published March 12, 2012
Although accurate quantitative amounts cannot be obtained as a
result of the complications that oxidized Pdi1p contains a partially
reduced domain and also nonlinear detection by immunoblot analy­
sis, we found that when the fraction of reduced Pdi1p was >0.8,
most Ero1pc-C100A-C105A was fully reduced, whereas when the
fraction of reduced Pdi1p was <0.3, most Ero1pc-C100A-C105A
was oxidized (Fig. 3 C). In a steady state, in vivo Pdi1p is partially
reduced (Vitu et al., 2010) like air-oxidized recombinant Pdi1p,
and almost all Ero1p is in a fully oxidized form (see 0 time point of
Figs. 1 B and 5 E). Therefore, we could conclude that partially
reduced Ero1pc is at least partially active and will continue to
oxidize Pdi1p until a ratio of oxidized-to-reduced Pdi1p is achieved
that no longer facilitates reduction and activation of Ero1p.
An Ero1p regulatory mutant is rapidly
activated by Pdi1p
Previously, we showed that an Ero1p mutant that lacks only one
of the three regulatory bonds, Ero1p(c)-C150A-C295A, has high
enzymatic activity both in vivo and in vitro (Sevier et al., 2007;
Vitu et al., 2010). This suggests that in the absence of the C150C295 regulatory bond, Ero1p is either more readily activated by
reduced Pdi1p or less easily inactivated by reaction with oxidized
Pdi1p or by autonomous oxidation.
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JCB • VOLUME 196 • NUMBER 6 • 2012
First, the autonomous oxidation of Ero1p regulatory
bond mutants Ero1pc-C150A-C295A, Ero1pc-C90A-349A, and
Ero1pc-C143A-166A was evaluated for their ability to undergo
autonomous reoxidation after being reduced by Trx1. This com­
parison is complicated by differing band mobility for the oxidized
and partially oxidized states of each of the mutants (Fig. 4 A).
As a measure of the oxidation rate that could be consistently
applied to each of the mutants, we represented the progression
of the oxidation process as the decline in the fraction of Ero1p
that was in a fully reduced state. All regulatory mutants showed
similar overall autonomous oxidation rates (Fig. 4 B), indicat­
ing that formation of the C150-C295 bond does not significantly
affect oxidation of other regulatory bonds. Because the pattern
of partially oxidized states was more complex for wild type
(Fig. 2 B) than for the mutants, it was not possible to make a
precise quantitative comparison of rates.
Next, we tested each of the three regulatory bond mutants
for their ability to be activated by reduced Pdi1p. This test re­
quired addition of the shuttle cysteine mutant to each of the three
regulatory bond mutants. Reduction of Ero1pc-C100A-C105AC150A-C295A by reduced Pdi1p was significantly faster than for
Ero1pc-C100A-C105A or for the other regulatory bond mutants
(Fig. 4, C and D). Ero1pc-C100A-C105A-C90A-C349A also
Downloaded from jcb.rupress.org on April 18, 2012
Figure 4. Mutational analysis of the roles of individual regulatory bonds in autonomous reoxidation of Ero1p. (A) Ero1pc-C150A-C295A, Ero1pc-C90AC349A, or Ero1pc-C143-C166A was reduced (red) by incubation with reduced Trx1 followed by Trx1 depletion and the oxidation (ox) state analysis.
(B) The fraction of reduced Ero1p was calculated by the ratio of the band intensity of reduced Ero1p species to the sum of fully reduced and fully oxidized
band intensities. (C) Mutational analysis of the roles of individual regulatory bonds in reduction by reduced Pdi1p of Ero1pc-C100A-C105A. The respective mutants were incubated with a 20-fold excess of reduced Pdi1p followed by the oxidation state analysis (the result of Ero1pc-C100A-C105A was
reproduced from Fig. 3 B). (D) The fraction of reduced Ero1p determined as in B reveals that the greatest rate enhancement occurs as a consequence of
elimination by mutation of the C150-C295 disulfide bond.
Published March 12, 2012
exhibited slightly faster reduction than Ero1pc-C100A-C105A or
Ero1pc-C100A-C105A-C143A-C166A (Fig. 4, C and D). These
results suggest that removal of the C150-C295 regulatory bond
lowers the threshold for Ero1p activation. Interestingly, both
the Ero1pc-C100A-C105A-C150A-C295A and Ero1pc-C100AC105A-C90A-C349A mutants exhibited some reoxidation of
Ero1p (Fig. 4 C) and oxidation of Pdi1p (not depicted) at the
60-min time point, suggesting that Ero1p may have alternative
pathways to relay disulfide bonds to Pdi1p when the shuttle disul­
fide is unavailable (see Discussion).
Pdi1p is the primary in vivo regulator
of Ero1p
If Pdi1p regulates Ero1p through reduction and oxidation of its
regulatory bonds, it should be possible to trap mixed disulfide
complexes between Pdi1p and the regulatory cysteines of Ero1p
in vivo. To specifically isolate mixed disulfides with regulatory
cysteines, we used FLAG epitope–tagged Ero1p-C100A-C105A
(expressed ectopically in cells that carry wild-type ERO1). Ero1pC100A-C105A is unable to support yeast viability as a result of
the disruption of its ability to oxidize Pdi1p in what has been
shown to be the primary pathway for electron exchange be­
tween Pdi1p and Ero1p in living cells (Frand and Kaiser, 2000).
Therefore, any observable mixed disulfides trapped between
Ero1p-C100A-C105A and Pdi1p in living cells would likely
represent thiol/disulfide exchange between the Ero1p regula­
tory cysteines and Pdi1p.
Ero1p-C100A-C105A-FLAG, isolated from cysteinealkylated cell extracts by immunoprecipitation with FLAG
antibody, formed a predominant mixed disulfide complex that
also reacted with Pdi1p antiserum (Fig. 5 A, lane 2). Analysis of
Downloaded from jcb.rupress.org on April 18, 2012
Figure 5. Pdi1p-mediated Ero1 regulation
in vivo. (A) Pdi1p forms mixed disulfide with
the Ero1p regulatory cysteines. Ero1p-C100AC105A-Flag and its variants were immunoprecipitated (IP) by anti-flag antibody and analyzed
by immuno­blots against anti-flag antibody and
anti-Pdi1p serum. Lane 1, Ero1p-C100A-C105AMyc; lane 2, Ero1p-C100A-C105A-flag; lane 3,
Ero1p-C100A-C105A-C90A-C349A-flag; lane 4,
Ero1p-C100A-C105A-C143A-C166A-flag; lane
5, Ero1p-C100A-C105A-C150A-C295A-flag. The
single asterisk indicates a monomer of Ero1p mutants, and the double asterisks indicate mixed disulfides between Ero1p mutants and Pdi1p. WB,
Western blot. (B) Pdi1p was depleted by growing
pdi1 covered with PGAL1-PDI1 in SMM (glucose
medium) for 15 h. (C) Immature CPY accumulates
as Pdi1p is depleted. A control for fully reduced
environment was prepared by treating cells with 1
mM DTT for 1 h. m, mature CPY; p1/p2, nascent
and glycosylated CPY, respectively. (D) Oxidation
(ox) states of Ero1p after Pdi1p depletion (glucose
medium) in a genetic background with pdi1 or
with pdi1 with deletions of all PDI homologs (PDI
homolog-). red, reduced. (E) The roles of Pdi1p or
glutathione on in vivo activation of Ero1p. Pdi1pdepleted cells (middle) and glutathione-depleted
cells (right) were prepared by growing CKY1081
in glucose medium and in galactose medium with
5 mM BSO, respectively. Cells were treated with
2 mM DTT for the indicated time.
the crude immunoprecipitate by mass spectroscopy identified
Pdi1p peptides but failed to identify additional PDI family
members, including Eps1p, Eug1p, Mpd1p, or Mpd2p (unpub­
lished data). This result suggests that Pdi1p is the primary oxi­
doreductase engaged in oxidation or reduction of the Ero1p
regulatory bonds. Loss of any single regulatory cysteine pair in
Ero1p-C100A-C105A did not prevent trapping of a mixed di­
sulfide bond between Ero1p-C100A-C105A and Pdi1p, indicat­
ing that Pdi1p interaction with Ero1p-C100A-C105A is not
limited to a single regulatory cysteine pair (Fig. 5 A). The mixed
disulfides between Pdi1p and the various Ero1p-C100A-C105A
regulatory bond mutants showed distinct mobilities on SDSPAGE (Fig. 5 A), which likely corresponded to different vari­
able combinations of cysteines in Ero1p and Pdi1p that engaged
in oxidation and reduction of Ero1p regulatory bonds.
If reduced Pdi1p were essential for activating Ero1p, de­
pletion of Pdi1p from the ER would be expected to decrease
activation of Ero1p by reduction of regulatory bonds even under
prevailing reducing conditions in the ER. We have established
conditions to deplete a cell of Pdi1p, which is essential for via­
bility, by glucose repression for 15 h of PGAL1-PDI1 in a pdi1
genetic background (Fig. 5 B; Tachibana and Stevens, 1992;
Frand and Kaiser, 1999). As expected, Pdi1p depletion disrupts
folding in the ER, as shown by the accumulation of the ER form
of carboxypeptidase Y (CPY), similar to that in cells that had
been treated with DTT (Fig. 5 C). Radiolabeling of cells to spe­
cifically follow CPY synthesized after Pdi1p depletion confirmed
a complete block of biosynthetic disulfide bond formation in
newly synthesized CPY (Fig. S4). However, under the same condi­
tions of Pdi1p depletion, most Ero1p remains oxidized (Fig. 5 D),
indicating that the signal for an insufficiently oxidized ER
Redox control of Ero1 • Kim et al.
719
Published March 12, 2012
cannot be transduced to activate Ero1p in the absence of Pdi1p.
Pdi1p-depleted cells showed very slow activation of Ero1p even
when they were treated with the reducing agent DTT, a condi­
tion that leads to rapid activation of Ero1p in Pdi1p-expressing
cells (Fig. 5 E).
In the Pdi1p-depleted cells, a small amount of activated (re­
duced) Ero1p was detected (Fig. 5 D), and catalytically inactive
Ero1 mutants Ero1p-C355S and Ero1p-C100A-C105A exhibited
significantly more of the reduced forms (Fig. S5), suggesting that
under conditions of Pdi1p depletion, although Ero1p can be re­
duced to some extent, most of the Ero1p remains in an oxidized
state if autonomous reoxidation is possible. We tested the possi­
bility that the residual reduction of Ero1p in cells that had been
depleted for Pdi1p was catalyzed by another PDI homolog. By
conducting the depletion experiment in a strain depleted for the
other homologs (mpd1, mpd2, eug1, eps1, pdi1, and
CEN-PGAL1-PDI1), we found that reduced Ero1p formed to at
least the same extent as in a pdi1 PGAL1-PDI1 strain (Figs. 5 D
and S5), indicating that no other PDI family members contribute
significantly to reduction of Ero1p regulatory bonds.
Glutathione acts synergistically with Pdi1p
to activate Ero1p
The rapid Pdi1p-dependent reduction of Ero1p regulatory bonds
that occurs in cells treated with DTT could be the result of a
pool of reduced Pdi1p formed by direct reduction with DTT that
had entered the ER. Alternatively, DTT treatment may produce
720
JCB • VOLUME 196 • NUMBER 6 • 2012
a pool of reduced glutathione (GSH) in the ER, which in turn
could lead to reduction of Pdi1p. To test the possibility that
DTT acts indirectly through glutathione, we tested the effect of
depleting cells of glutathione by treating cells with 5 mM buthi­
onine sulfoximine (BSO) for 15 h, which blocks glutathione
synthesis by inhibiting -glutamylcysteine synthetase. Reduction
of Ero1p regulatory bonds by DTT treatment occurred more slowly
in glutathione-depleted cells than in untreated cells (Fig. 5 E).
This result suggests that the presence of a reducible pool of glu­
tathione may accelerate the overall process of reduction of Pdi1p
by DTT. Alternatively, depletion of glutathione by BSO treat­
ment may subtly affect Pdi1p expression or have some other
indirect effect on the redox state of the ER.
To address the role of glutathione in Ero1p activation more
directly, we reconstituted Ero1p activation in vitro using both
Pdi1p and GSH as substrates. By itself, Pdi1p is a poor activator of
Ero1p because once Pdi1p has become oxidized through Ero1p
oxidase activity, there is no longer sufficient reduced Pdi1p avail­
able to keep Ero1p in an active state, and oxidized Pdi1p can
accelerate inactivation of Ero1p. Therefore, fully activated Ero1p
was rarely observed during reaction with reduced Pdi1p (Fig. 6 A).
Glutathione alone was also a poor activator of Ero1p. 10 mM
GSH did not reduce regulatory bonds in either wild-type Ero1pc
(Fig. 6 B, left) or Ero1pc-C100A-C105A (not depicted). However,
a mixture of 10 mM GSH and Pdi1p maintained a pool of at
least partially activated Ero1p (Fig. 6 B, right). The basis of Ero1p
activation in the presence of Pdi1p and GSH appeared to be that
Downloaded from jcb.rupress.org on April 18, 2012
Figure 6. A coupled Pdi1p/GSH system recapitulates Ero1p homeostasis in vitro. (A) Ero1p is only partially activated when Pdi1p is used as a substrate.
1 µM Ero1pc was incubated with 25 µM reduced (red) Pdi1p. Ero1pc and Pdi1p were detected by immunoblotting with anti-Ero1p serum or anti-HDEL
antibody. At the endpoint of in vitro oxidation (ox), Pdi1p is in a partially oxidized state, indicated by ox*. (B) Pdi1p is necessary for efficient reduction of
Ero1pc by GSH. Ero1pc or a mixture of Ero1pc and oxidized Pdi1p was mixed with different concentrations of GSH for 1 h followed by TCA precipitation, NEM modification, and Western analysis. (C) Pdi1p is necessary for efficient oxidation of GSH by Ero1pc. 10 mM GSH was incubated with 1 µM
Ero1pc in the presence or absence of 10 µM oxidized Pdi1p. The amount of GSSG produced was measured after alkylating GSH followed by reduction
with glutathione reductase and assay of free thiols with Ellman’s reagent. (D) The balance of GSH and GSSG can be established by the mixture of Ero1pc
and Pdi1p. 10 µM Pdi1p and 5 µM Ero1pc were incubated with three different glutathione mixtures, 5 mM GSH, 4 mM GSH/0.5 mM GSSG, and 3 mM
GSH/1 mM GSSG. The natural oxidation of 5 mM GSH without enzymes was also measured. The percentage of oxidized glutathione was calculated for
the indicated time points. (C and D) Error bars represent the SEM generated by three independent experiments.
Published March 12, 2012
Discussion
A conserved protein disulfide relay system of the ER, consist­
ing of Ero1 and PDI, supports correct disulfide bond formation
of secretory proteins (Sevier et al., 2001; Sevier and Kaiser,
2006a; Appenzeller-Herzog et al., 2008; Braakman, 2009). In
S. cerevisiae, the Ero1-PDI relay is essential for biosynthetic
disulfide bond formation and is thus essential for cell viability
(Frand and Kaiser, 1998; Pollard et al., 1998; Vitu et al., 2010).
We previously showed that the activity of S. cerevisiae Ero1p is
controlled by an autoregulatory feedback loop that involves the
reduction and the reoxidation of three regulatory disulfide bonds
(Sevier et al., 2007). Here, we show that not only is Pdi1p the
major substrate of Ero1p in the disulfide relay system but that
Pdi1p is also the primary regulator of Ero1p activity by its abil­
ity carry out dithiol/disulfide exchange with the regulatory
bonds. We also show that the redox state of glutathione in the
ER is important for setting the regulatory state of Pdi1p, and, in
this way, the disulfide bond–generating activity of Ero1p is con­
trolled by the redox status of both Pdi1p and of glutathione.
In a reaction initiated by adding to Ero1p a stoichiometric
excess of reduced Trx1, a model substrate, the regulatory bonds
in Ero1p are rapidly reduced, and Trx1 is oxidized by active Ero1p.
Once Trx1 has been oxidized, the regulatory bonds in Ero1p are
reformed, and Ero1p returns to an inactive state. In this paper,
we separated the activation and inactivation phases of this cycle
to determine the biochemical requirements for each phase.
As expected, the reduced form of Trx1 readily reduces regula­
tory bonds in Ero1p and is thus a potent activator of Ero1p.
However, oxidized Trx1 has no capacity to reoxidize the regu­
latory cysteines of Ero1p. Instead, oxidation of the regulatory
cysteines is accomplished by Ero1p itself in a reaction that
probably involves the transfer of a disulfide bond from the shut­
tle cysteines of one Ero1p protein molecule to the regulatory
cysteines of another. Thus, the inactivation phase occurs by
autonomous oxidation of Ero1p once reduced Trx1 has been
depleted by being converted to the oxidized form.
A similar analysis for a reaction that contains Ero1p and
its natural substrate Pdi1p revealed that Ero1p is never fully ac­
tivated in the presence of Pdi1p (Fig. 6 A). Whereas we initially
thought that the inability of Pdi1p to activate Ero1p robustly
was a consequence of some deficiency in the in vitro reaction,
we now have evidence that the balance of the reduced and
oxidized forms of Pdi1p is crucial in regulating Ero1p enzyme
activity and faithfully reproduces the redox homeostasis set
point of the ER.
Pdi1p has two redox-active domains containing the CxxC
motifs. The a domain is not as efficiently oxidized by Ero1p as
the a domain (Vitu et al., 2010). However, the a domain does
appear to play a greater role than the a domain in oxidizing the
Ero1p regulatory bonds; the partially oxidized a domain (Pdi1pSSCC) accelerates oxidation of Ero1p regulatory bonds more rap­
idly than the fully oxidized a domain (Pdi1p-CCSS). Given the
relative redox potentials of the two domains, their propensity to be
oxidized by Ero1p and their relative ability to oxidize the regu­
latory bonds of Ero1p provide a consistent explanation for why
the a domain is only partly oxidized, whereas the a domain is
fully oxidized in cells under standard conditions (Vitu et al.,
2010). In contrast to the yeast Pdi1p, the two active domains of
the human PDI have similar reduction potentials, and the a
domain is the one preferentially oxidized by Ero1- (Wang et al.,
2009; Chambers et al., 2010). Therefore, it seems possible that
the mechanism of Ero1- regulation via the two domains of the
human PDI is distinct from the one in yeast. Specificity in inter­
action between human and yeast Ero1 and PDI proteins has
been demonstrated (Vitu et al., 2010; Masui et al., 2011). Com­
parison of the activity of human and yeast proteins in vitro
shows that the highest enzyme activity is achieved using protein
combinations of Ero1 and PDI from the same species (Araki
and Nagata, 2011). Low catalytic activity was traced in part to
a weaker binding affinity between human Ero1- and yeast Pdi1p,
relative to human PDI (Araki and Nagata, 2011). Whether the
different redox potentials of the PDI domains in human and
yeast PDI also contribute to the differences in Ero1 regulation
between species remains to be assessed.
Several lines of evidence point to the existence of multiple
disulfide relay pathways from Ero1p to Pdi1p. Best character­
ized is the flow of disulfides from the C352-C355 pair to Pdi1p
via the C100-C105 shuttle cysteines. This pathway is necessary
for full activity of Ero1pc in vitro and is necessary for cell via­
bility. Nevertheless, Ero1pc-C100A-C105A retains some activ­
ity in vitro and shows slow autonomous reoxidation, showing
that a disulfide bond can be transferred, albeit slowly, from the
active site to the regulatory disulfides without the action of the
C100-C105 shuttle (Fig. 2 B). Moreover, in vivo experiments
have shown that yeast cells with an ero1-C100A mutation are
Redox control of Ero1 • Kim et al.
Downloaded from jcb.rupress.org on April 18, 2012
provided by a pool of GSH in great stoichiometric excess to Pdi1p,
which allowed for a significant pool of reduced Pdi1p to be main­
tained. This pool of reduced Pdi1p in turn could reduce the regu­
latory bonds of Ero1p, thus overcoming the kinetic barrier for
GSH to reduce the Ero1p regulatory bonds. In this coupled reac­
tion, oxidized glutathione (GSSG) was generated by the com­
bined action of Pdi1p and Ero1p (Fig. 6 C), and production of
GSSG depended on Ero1p enzymatic activity because even in the
presence of Pdi1p, catalytically inactive Ero1pc-C100A-C105A
did not produce GSSG actively, whereas hyperactive Ero1pcC150A-C295A produced GSSG more rapidly than wild type
(not depicted).
To investigate the effects of the balance of GSH to GSSG on
Ero1p activity, we reconstituted an in vitro reaction in which re­
combinant Ero1p and Pdi1p were incubated in the presence of
three different ratios of GSH/GSSG. When the reaction was be­
gun in the presence of GSH only, the rate of glutathione oxidation
was initially rapid but then slowed. Evidently, the concomitant
generation of GSSG by the action of Ero1p and Pdi1p eventually
leads to inactivation of Ero1p (Fig. 6 D, 5 mM GSH curve). Intro­
duction of GSSG into the coupled reaction slowed the initial rate
of GSH oxidation (compare the curves of 5 mM GSH with 8:1 and
3:1 GSH/GSSG; Fig. 6 D). Taking the background of slow nonen­
zymatic generation of GSSG into account, the Ero1p and Pdi1p
catalyzed oxidation of GSH stops at when the molar ratio of GSH/
GSSG is 3:1. Thus, this ratio of GSH/GSSG appears to be the
approximate steady-state set point for the coupled reaction.
721
Published March 12, 2012
722
JCB • VOLUME 196 • NUMBER 6 • 2012
Figure 7. A balance between reduced and oxidized Pdi1p determines
ER redox homeostasis. The thiol substrates (reduced protein thiols or reduced glutathione) present in the ER can be oxidized by Pdi1p, leading
to formation of reduced Pdi1p. If sufficient reduced Pdi1p has formed,
Ero1p is activated by direct reduction of the Ero1p regulatory bonds by
reduced Pdi1p. Once activated, Ero1p oxidizes Pdi1p until a steady-state
balance of oxidized and reduced Pdi1p is reestablished. The balance of
reduced and oxidized Pdi1p determines the degree of Ero1p activation;
once reduced Pdi1p has declined sufficiently, autonomous oxidation of
the regulatory bonds of Ero1p will lead to Ero1p inactivation, preventing
overoxidation of the ER. Under hyperoxidizing conditions, oxidized Pdi1p
would rapidly inactivate Ero1p.
(Appenzeller-Herzog et al., 2010). It seems likely that ER redox
homeostasis is maintained by the same basic mechanism in yeast
and mammalian cells.
Materials and methods
Protein expression and purification
The sequence coding Ero1pc (56–424) or Ero1pc mutants (Ero1pc-C100AC105A, Ero1pc-C355S, Ero1pc-C150A-C295A, Ero1pc-C90A-C349A,
Ero1pc-C143A-C166A, and Ero1pc-C100A-C105A-C143A-C166A) was
fused at the C terminus to His6-tagged MBP linked by the Tobacco etch virus
(TEV) protease cleavage sequence (ENLYFQGS) inserted into the pET17b
vector (EMD) between NdeI and XhoI restriction sites. We found that C355S
was the only point mutation of the active-site cysteine pair (C352-C355)
that could be produced in a soluble FAD-bound state. Ero1pc and all variants were overexpressed in Origami B(DE3) competent cells (EMD) by induction with 0.4 mM IPTG supplemented with 10 µM FAD overnight at
room temperature. Ero1pc fusion proteins with His6-tagged MBP were purified from cell lysates by affinity to amylose (New England Biolabs, Inc.)
and then dialyzed into TEV cleavage buffer (50 mM Tris, pH 7.8, 0.5 mM
EDTA, and 0.5 mM tris(2-carboxyethyl)phosphine). MBP-fused Ero1pc
(MBP-Ero1pc) and Ero1pc-C100A-C105A were also expressed and purified as described for Ero1pc purification. Ero1pc was cleaved from His6tagged MBP by incubating with recombinant His6-tagged TEV protease
(1:100 wt/wt protease to substrate; the TEV protease–encoding plasmid
was provided by C. Hill, University of Utah, Salt Lake City, UT) for 15 h at
25°C. Ero1pc and variants cleaved from His6-MBP were collected by flowing through the HiTrap Ni2+ column (GE Healthcare) to remove uncleaved
fusion protein, cleaved His6-tagged MBP, and TEV protease. Some Ero1pc
variants (Ero1pc-C100A-C105A-C150A-C295A and Ero1pc-C100AC105A-C90A-C349A) were subcloned in pGEX-4T vector purified by flowing though GSTrap (GE Heathcare) followed by thrombin cleavage for
elution and secondary purification with the HiTrap Ni2+ column for removal
of thrombin (Gross et al., 2004). All purified Ero1pc proteins were dialyzed against PBS.
The sequence coding Pdi1p (31–522) or Pdi1p variants (C406SC409S for CCSS, C61S-C64S for SSCC, and C61S-C64S-C406S-C409S
Downloaded from jcb.rupress.org on April 18, 2012
viable if they have the capacity to up-regulate Ero1p expression
through unfolded protein response induction (Frand and Kaiser,
2000), and overexpression of Ero1p-C100A-C105A-C150AC295A was able to partially suppress the temperature sensitiv­
ity of the ero1-1 strain (unpublished data).
The pathway of dithiol/disulfide exchange reactions used
by Ero1p-C100A-C105A to oxidize Pdi1p remains to be deter­
mined. Considering that autonomous oxidation of the regulatory
cysteines by Ero1p-C100A-C105A is possible, we hypothesize
that the active-site disulfide bond C352-355 may first be trans­
ferred to regulatory cysteine pairs, and then Pdi1p could be oxi­
dized by reducing these regulatory bonds during the activation
process of Ero1p. These pathways were revealed by experi­
ments with Ero1pc-C100A-C105A but are not limited to mutant
Ero1p and are likely also used by wild-type Ero1p. By transfer­
ring one or two of the regulatory bonds to Pdi1p, Ero1p would
have some capacity to oxidize a stoichiometric quantity of Pdi1p
without any Ero1p becoming fully active. The C143-C166 bond
may be the most labile regulatory bond, as its reduction is the
first step of Ero1 activation by Pdi1p in vitro (Heldman et al.,
2010) and in turn could readily contribute to Pdi1p oxidation.
This pathway may allow the disulfide relay system to respond
to modest increases in load with a minimum of excess hydrogen
peroxide generation.
When Ero1p is incubated with reduced Pdi1p and GSH,
Ero1p can be shown to be catalytically active by its ability to oxi­
dize a stoichiometric excess of GSH, although little or no Ero1p
can be detected in a state in which all regulatory cysteines are in
a reduced state (Fig. 6 B). As previously noted (Heldman et al.,
2010), this observation indicates that the partially reduced forms
of Ero1p have at least some oxidase activity. Thus, the regula­
tory bonds in Ero1p may act less like an on/off switch than a
proportional controller with different degrees of activity accord­
ing to the redox states of the three regulatory bonds. We have
not been able to devise a quantitative test of this attractive hy­
pothesis because it is not possible to isolate in pure form Ero1p
in a partially reduced state.
Toward a biochemical understanding of the mechanism of
Ero1p regulation in a highly simplified reaction, we success­
fully reconstituted the disulfide relay system and the basic prop­
erties of redox homeostasis in vitro with just their components,
Ero1pc, Pdi1p, and glutathione (Fig. 6). The capacity of Pdi1p
to sustain Ero1p in an activated state can be increased by addi­
tion of an excess of GSH in the in vitro reaction. This coupled
reaction appears to approximate conditions in the ER (Hwang
et al., 1992), in which transfer of disulfide bonds to nascent se­
cretory proteins and to GSH keeps enough Pdi1p in a reduced
state to allow for some capacity to activate Ero1p. In the cou­
pled reaction, Ero1p enzyme activity is controlled by the ratio
of GSH/GSSG, and a 3:1 ratio of GSH/GSSG appears to be close
to the steady-state set point at which Ero1p can be activated.
Pdi1p, which has both reductase and isomerase activities, can
serve as the key link between thiol substrates, including gluta­
thione, and Ero1p to activate the disulfide relay system only
when substrates are present (Fig. 7). In the mammalian cell, it
has also been suggested that oxidation of PDI and glutathione
is well regulated by an equilibrium between Ero1- and PDI
Published March 12, 2012
for SSSS) tagged with His6 at the N terminus was inserted into pET20b between NdeI and SacI restriction sites. Recombinant His6-tagged Pdi1p,
Pdi1p variants, and His6-tagged E. coli thioredoxin (Trx1; Sevier and Kaiser,
2006b) were overexpressed in BL21(DE3)pLysS competent cells (EMD) by
induction with 0.4 mM IPTG for 5 h at 37°C and purified from cell lysates
by affinity to the HiTrap Ni2+ column. Purified, naturally oxidized Pdi1p
and Trx1 were dialyzed into PBS. Reduced forms of Pdi1p and Trx1 were
prepared by incubating with 10 mM DTT for 30 min at room temperature
and removing DTT with Micro Bio-Spin 6 (Bio-Rad Laboratories).
Analysis of protein oxidation states
For reduction of Ero1p regulatory bonds in vitro, 1 µM Ero1pc or its variants
were incubated with 50 or 20 µM reduced Trx1, reduced Pdi1p, or reduced
Pdi1p mutants in PBS containing 0.5 mM EDTA. After incubation at 25°C,
the reactions were quenched by mixing with 1× SDS-AMS (4-acetamido4-maleimidylstilbene-2,2-disulfonic acid) buffer (1× SDS sample buffer
without reducing agent and 1 mM AMS [Invitrogen]), which alkylates free
cysteine thiols and adds one 500-D moiety to each free thiol. For reactions that contained glutathione or DTT, proteins were first precipitated by
10% chilled TCA. Protein precipitants were washed with 0°C acetone and
were redissolved with 1× SDS-AMS buffer. Ero1p and Pdi1p were analyzed by nonreducing SDS-PAGE followed by immunoblotting with antiEro1p serum or anti-HDEL (Santa Cruz Biotechnology, Inc.). The oxidation
state of Trx1 was analyzed by nonreducing SDS-PAGE followed by staining with SimplyBlue SafeStain (Invitrogen).
The in vivo oxidation state of Ero1p was assessed in cells growing
in SMM medium incubated with 0, 0.2, or 1 mM DTT for 30 min and then
lysed in the presence of chilled 10% TCA by agitating with glass beads.
Protein pellets from cell lysates were washed with chilled acetone and redissolved with 1× SDS-AMS buffer. Oxidation states of Ero1p and variants
were analyzed by nonreducing SDS-PAGE and immunoblotting with antiEro1p. Immunoblots were visualized with an Odyssey imaging system (LI-COR
Biosciences). The fully reduced or the oxidized band of Ero1pc was quantified
by the Odyssey program (v2.0; LI-COR Biosciences), and reduced fraction
Downloaded from jcb.rupress.org on April 18, 2012
Yeast strains and plasmids
CKY1044 and CKY1045 and MATa and MAT pdi1::KanMX strains
covered by URA3 CEN PDI1 plasmid pCS213 have been previously described (Vitu et al., 2010). To facilitate scoring of crosses with the pdi1
strain, a NatMX-marked pdi1 strain was constructed by swapping the
KanMX marker in CKY1045 with a NatMX4 cassette by homologous recombination (Goldstein and McCusker, 1999). Individual disruptions of the
genes encoding the yeast PDI homolog proteins were made by one-step
gene replacement of the entire ORF with KanMX by homologous recombination. The compound deletion strain CKY1086 with the genotype MATa GAL2
ura3-52 leu2-3,112 pdi1::NatMX mpd1::KanMX mpd2::KanMX
eug1::KanMX eps1::KanMX [pCS213] was constructed by sequential
rounds of mating, sporulation, and scoring of the initial individual disruption
mutant strains. Disruption of all five genes encoding the PDI1 homologs in
CKY1086 was confirmed by PCR analysis. Some pdi1 strains also contained a genomic-tagged copy of BGL2. Tagging of BGL2 with a triple HA
epitope at its C terminus was performed by homologous recombination with
a 3HA-KanMX6 module (Longtine et al., 1998). To construct the MATa GAL2
ura3-52 leu2-3,112 pdi1::NatMX BLG2:HA-KanMX [pCS213] double
mutant (CKY1087), individual pdi1 and BLG2:HA strains were mated,
and an MATa NatMX+ KanMX+ segregant was selected after sporulation.
Viability of CKY1086 and CKY1087 depends on a plasmid-borne PDI1 gene
provided by plasmid pCS213. CKY1088 and CKY1089 were generated
by transformation of CKY1086 or CKY1087, respectively, with the pSK65
[CEN LEU2 PGAL1-PDI1] followed by selection against pCS213 by plating on
supplemented minimal medium (SMM) with 5-FOA. CKY1090 was created
by transformation of CKY1044 with pSK2 [CEN LEU2 His6-PDI1] followed
by selection against pCS213 with 5-FOA.
To create pSK2 [CEN LEU2 His6-PDI1], the sequence coding HHHHHGG was inserted right after the signal sequence (1–22) by introducing
the NheI restriction site in pCS463, which contains the PDI1 coding region
and 875 and 150 bp of the 5 and 3 untranslated regions. Plasmid
pSK65 [CEN LEU2 PGAL1-PDI1] contains the PDI1 coding region and 60
and 150 bp of the 5 and 3 untranslated regions under the GAL1 promoter. ERO1-coding URA3 2µ plasmids pSK63 [ERO1-flag], pSK64 [ero1C100A-C105A-flag], pDPS51 [ero1-C100A-C105A-C150A-C295A-flag],
pDPS49 [ero1-C100A-C105A-C90A-C340A], pDPS50 [ero1-C100AC105A-C143A-C166A], and pSK66 [ero1-C355S-flag] were generated by
insertion of sequence-encoding flag tag right after the last codon using the
NotI restriction site and subcloning or site-directed mutagenesis based on
the previously reported plasmid pAF84 (Frand and Kaiser, 1998).
was calculated by dividing reduced band intensity with the sum of reduced
and oxidized band intensity.
To determine the effect of hydrogen peroxide on the oxidation state of
Ero1p regulatory bonds (Fig. S1), 1 µM Ero1pc was incubated with 100 µM
reduced His6-tagged Trx1 in PBS containing 0.5 mM EDTA for 2 min, and
Trx1 was selectively removed by affinity for Ni2+ beads. To remove hydrogen peroxide generated during action of Ero1p oxidase, 80 µg/ml catalase was added to the reaction. Removal of hydrogen peroxide by catalase
was confirmed by measuring the amount of hydrogen peroxide in the reaction with Amplex UltraRed reagent (Invitrogen). 1 µM Ero1pc was incubated with 100 µM reduced thioredoxin, 50 µM Amplex red, and 0.1 U/ml
HRP in the presence or absence of 80 µg/ml catalase for 30 min at room
temperature. Fluorescence was measured in a microplate reader with
wavelength settings of 530/590 nm. Inactivation of Ero1p under anaerobic conditions was analyzed as described above except being performed
in the anaerobic chamber flowing N2 and CO2 gases. To test whether
exogenous hydrogen peroxide or oxidized Pdi1p can promote inactivation
of Ero1p, 100 µM hydrogen peroxide or 20 µM oxidized Pdi1p was
added to Ero1pc, Ero1pc-C100A-C105A, or Ero1pc-C355S after Trx1 depletion. Oxidation states of Ero1pc and its mutants were analyzed after
cysteine modification with 1 mM AMS by nonreducing SDS-PAGE and
immunoblotting with anti-Ero1p serum.
The oxidation states of each catalytic domain of recombinant Pdi1p
were determined by introducing a thrombin cleavage site that allows separation of the two catalytic domains (Fig. S2). The sequence coding N-terminal
His6-tagged Pdi1p (31–522) with a thrombin recognition sequence in the
X-linker was subcloned from pSK42 (Vitu et al., 2010) for E. coli expression. His6-Pdi1p-thrombin was overexpressed in BL21(DE3)pLysS competent cells by IPTG and purified from cell lysates with the HiTrap Ni2+ column.
Purified His6-Pdi1p-thrombin was dialyzed into PBS. 10 µg His6-Pdi1p-thrombin
was incubated with 1 mM AMS for 30 min at room temperature followed
by treatment with 0.1 µg thrombin supplemented with 1 mM CaCl2 for 1 h
at 37°C. Fully reduced Pdi1p control was prepared by treatment with
10 mM DTT for 30 min at room temperature. DTT was removed by using
Bio-Spin 6. AMS-modified and thrombin-digested samples were analyzed
by nonreducing SDS-PAGE.
The oxidation state of newly synthesized CPY in the Pdi1p-depleted
ER was analyzed in pdi1 cells with galactose-inducible PDI1 grown to the
exponential phase for 15 h in glucose medium to deplete Pdi1p. Cells
were suspended in SMM-met, pulse labeled for 10 min at 30°C, and lysed
in the presence of 10% chilled TCA to block disulfide exchange. After AMS
modification, CPY was immunoprecipitated and resolved by reducing
SDS-PAGE. A fully reduced CPY control was prepared by treating cells
with 10 mM DTT for 15 min before cell lysis.
Reoxidation of Ero1pc after thioredoxin depletion
1 µM Ero1pc was reduced by incubation with 100 µM reduced His6-tagged
Trx1 for 2 min and then passed through Ni2+ beads to remove Trx1. At
times after Trx1 depletion, the reaction was quenched by mixing with SDS
sample buffer containing 1 mM AMS. Depletion of Trx1 was confirmed by
analysis with nonreducing SDS-PAGE. When necessary, 40 µM oxidized
Trx1, 20 µM oxidized Pdi1p, or 20 µM oxidized Pdi1p mutant was added
into Ero1pc or Ero1pc mutants immediately after Trx1 depletion.
Purification of mixed disulfides from yeast
CKY1090 expressing His6-Pdi1p was transformed with pSK64 [ero1C100A-C105A-flag], pDPS51 [ero1-C100A-C105A-C150A-C295A-flag],
pDPS49 [ero1-C100A-C105A-C90A-C340A], and pDPS50 [ero1-C100AC105A-C143A-C166A]. Exponentially grown cells were lysed by agitation with glass beads in the presence of chilled 10% TCA. TCA-precipitated
protein pellets were washed with acetone and redissolved with 2% SDS and
50 mM Tris, pH 8.0, containing 50 mM NEM to block free cysteines. For efficient affinity purification, SDS was removed by SDS-Out reagent (Thermo
Fisher Scientific). After clarification by centrifugation, Ero1p-C100A-C105Aflag was released from anti-flag affinity beads using 100 µg/ml flag peptide (Sigma-Aldrich) followed by deglycosylation with PNGaseF for 3 h at
37°C. Samples purified by affinity to anti-flag beads were passed through
an Ni2+ column to capture mixed disulfides with His6-Pdi1p. Both Ni2+
unbound fraction and bound fraction were collected and analyzed by nonreducing SDS-PAGE and immunoblotting with anti-flag (Sigma-Aldrich) or
anti-Pdi1p.
Depletion of Pdi1p and glutathione in yeast
To determine the oxidation states of Ero1p and mutants in Pdi1p- or
glutathione-depleted cells, pSK63, pSK64, and pSK66 were transformed into
CKY1088 and/or CKY1089. Cells exponentially grown in SMM containing
Redox control of Ero1 • Kim et al.
723
Published March 12, 2012
1.5% galactose and 1% raffinose were incubated in SMM containing 2%
glucose for 15 h to shut off expression of Pdi1p. Glutathione was depleted
by growing cells in the presence of 5 mM BSO, an inhibitor of glutathione
biogenesis, for 15 h. Pdi1p depletion was confirmed by immunoblotting
with anti-Pdi1p (Fig. 5 E), and glutathione depletion was confirmed using
Ellman’s reagent.
The maturation of CPY was evaluated in pdi1 cells with galactoseinducible PDI1 grown to exponential phase for 15 h in glucose medium to
deplete Pdi1p. For fully reduced cell environment, cells were incubated
with 1 mM DTT for 1 h after a 14-h incubation in glucose medium. Cell lysates
was prepared in the presence of chilled 10% TCA, and mature and immature
CPY were analyzed by immunoblotting with anti-CPY serum.
Online supplemental material
Fig. S1 shows that autonomous oxidation and inactivation of Ero1p is not
a side effect of hydrogen peroxide produced by aerobic catalysis by
Ero1p. Fig. S2 shows that the endpoint of air oxidation yields a form of
recombinant Pdi1p with the a domain almost fully oxidized, and the a
domain is partially oxidized. Fig. S3 shows that air-oxidized Pdi1p can
contribute to the oxidation of the Ero1p regulatory bonds. Fig. S4 shows
that when cells are depleted of Pdi1p by growing pdi1 cells with glucoserepressible PDI1 in glucose medium, newly synthesized CPY remains largely
in a reduced state. Fig. S5 shows that when cells are depleted of Pdi1p,
autonomous oxidation of the regulatory bonds of Ero1p can occur. This
oxidation requires active-site cysteine C355 but does not require the shuttle
cysteines C100 and C105. Online supplemental material is available at
http://www.jcb.org/cgi/content/full/jcb.201110090/DC1.
The authors thank Debbie Fass for thoughtful suggestions.
This work was supported by a grant from the National Institutes of
Health (GM46941) to C.A. Kaiser.
Submitted: 20 October 2011
Accepted: 10 February 2012
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